General Background of Antibodies
Murine monoclonal antibodies are widely used as diagnostic and therapeutic agents in the treatment of human disease. Mice can be readily immunized with foreign antigens to produce a broad spectrum of high affinity antibodies. Invariably, the introduction of murine or other rodent antibodies into humans results in the production of a human anti-mouse antibody (HAMA) response due to the presentation of a foreign protein in the body. The production of HAMA in patients can result from the introduction of foreign antibody in a single dose or from extended use in therapy, for example, for the treatment of cancer. Extended use of murine antibody is generally limited to a term of days or weeks in patients before concerns of anaphylaxis arise. Moreover, once HAMA has developed in a patient, future use of murine antibodies for diagnostic or therapeutic purposes is often precluded for the same reasons.
Beyond ethical considerations, attempts to produce human monoclonal antibodies have not been highly successful for a number of reasons. The production in vitro of human monoclonals rarely results in high affinity antibodies. In vitro cultures of human lymphocytes yield a restricted range of antibody responses relative to the broad spectrum of reactive antibodies produced in vivo through direct immunization of mice. Additionally, in humans, immune tolerance prevents the successful generation of antibodies to self-antigens. All of these factors have contributed to the search for ways to modify the structures of murine monoclonal antibodies to improve their use in patients. Many investigators have attempted to alter, reshape or humanize murine monoclonal antibodies in an effort to improve the therapeutic application of these molecules in patients.
Strategies of Antibody Humanization
The earliest reports of the controlled rearrangement of antibody domains to create novel proteins was demonstrated using rabbit and human antibodies as described by Bobrzecka, K. et al. (Bobrzecka, K., Konieczny, L., Laidler, P. and Rybarska, J. (1980), Immunology Letters 2, pp. 151-155) and by Konieczny et al. (Konieczny, L., Bobrzecka, K., Laidler, P. and Rybarska, J. (1981), Haematologia 14 (I), pp. 95-99). In those reports, the protein subunits of antibodies, rabbit Fab fragments and human Fc fragments, were joined through protein disulfide bonds to form new, artificial protein molecules or chimetic antibodies.
Recombinant DNA technology was used to construct gene fusions between DNA sequences encoding mouse antibody variable light and heavy chain domains and human antibody light chain (LC) and heavy chain (HC) constant domains to permit expression of the first recombinant "near-human" antibody (chimeric antibody) product (Morrison, S. L., Johnson, M. J., Herzenberg, L. A. and Oi, V. T. (1984), Proc. Natl. Acad. Sci. U.S.A. 81, pp. 6851-6855).
The kinetics and immune response in man to chimeric antibodies has been examined (LoBuglio, A. F., Wheeler, R. H., Trang, J., Haynes, A., Rogers, K., Harvey, E. B., Sun, L., Ghrayeb, J. and Khazaeli, M. B. (1989), Proc. Natl. Acad. Sci. 86, pp. 4220-4224).
Chimeric antibodies contain a large number of non-human amino acid sequences and are immunogenic in man. The result is the production of human anti-chimera antibodies (HACA) in patients. HACA is directed against the murine V-region and can also be directed against the novel V-region/C-region (constant region) junctions present in recombinant chimeric antibodies.
To overcome some of the limitations presented by the immunogenicity of chimeric antibodies, the DNA sequences encoding the antigen binding portions or complementarity determining regions (CDR's) of murine monoclonal antibodies have been grafted by molecular means in the DNA sequences encoding the frameworks of human antibody heavy and light chains (Jones, P. T., Dear, P. H., Foote, J., Neuberger, M. S. and Winter, G. (1986), Nature 321, pp. 522-525; Riechmann, L., Clark, M., Waldmann, H. and Winter, G. (1988), Nature 332, pp. 323-327). The expressed recombinant products called reshaped or humanized antibodies are comprised of the framework of a human antibody light or heavy chain and the antigen recognition portions, CDR's, of a murine monoclonal antibody. Several patent applications have been filed in this area including, for example, European Patent Application, Publication No. 0239400; European Patent Application, Publication Nos. 0438310A1 and 0438310A2; International Patent Publication No. WO 91/09967; and International Patent Publication No. WO 90/07861.
However, it is questionable whether European Patent Application (EP), Publication No. 0239400 is truly enabling. It is not assured in this patent that the best fit is made to assure proper presentation of the CDR loops at the antibody combining site.
EP Publication Nos. 0438310A1 and 0438310A2 go a step beyond EP Publication No. 0239400 by protecting the importance of uniquely selected human frameworks for the human light chain (LC) and heavy chain (HC) V-regions. These V-region frameworks should show a high degree of sequence similarity with the frameworks of the murine monoclonal antibody and present the CDR's in the appropriate configuration. However, the criteria for sequence matching are no more sophisticated than simple homology searching of the antibody protein or DNA databases.
International Patent Publication No. WO 91/09967 attempts a further variation of the method disclosed in EP Publication No. 0239400. In International Patent Publication No. WO 91/09967, homology of the donor sequences and the acceptor framework is not important, rather it discloses that a selected set of residues in the LC and HC are critically important to humanization. The ability to make changes at these positions is the basis of International Patent Publication No. WO 91/09967.
International Patent Publication No. WO 90/07861 proposes four important criteria for designing humanized antibodies. 1) Homology between human acceptor and non-human donor sequences. 2) Use donor rather than acceptor amino acids where the acceptor amino acid is unusual at that position. 3) Use donor framework amino acids at positions adjacent to the CDR. 4) Use donor amino acids at framework positions where the sidechain atom is within 3 Angstroms of the CDR in a 3-D model. The first antibody humanized by this method retained less than 1/3 the affinity of the original monoclonal antibody.
None of the above methods for designing a humanized antibody is predictable due to the questions that surround CDR framework interactions. By replacement of murine framework with human framework, there is no guarantee of identical conformations for CDR's because i) the V.sub.L -V.sub.H interaction is not identical in all V-regions and ii) accurate prediction of the CDR-framework interactions are key to faithful reproduction of the antigen binding contacts.
The above methods do not offer a general solution to solving the issues surrounding antibody humanization, rather the methods as outlined in each reference above involve a substantial amount of trial and error searching to obtain the desired affinity in the final humanized product. More importantly, there is no guarantee that corrective changes in framework amino acids will leave the reshaped V-regions resembling the surface character of a truly human antibody. Therefore, it can be argued that antibodies humanized by the above methods may be immunogenic in man.
Antigenicity of Antibodies
The antigenicity/immunogenicity of an antibody, including recombinant reshaped antibody products, introduced into humans can be viewed as a surface phenomenon. In general one can view the immune system as scanning the surface of a protein introduced to the body. If the F.sub.V portion of a humanized antibody `opens-up` in the circulation then internal residues can be presented to the immune system. On the other hand, if the F.sub.V portion is stable and tightly packed then only the surface residues presented by the V-regions and the interface between the V.sub.L and V.sub.H regions will be `scanned`.
Surface Reshaping or Resurfacing of Antibodies
The notion of surface presentation of proteins to the immune system raises the prospect of redesigning murine monoclonal antibodies to resemble human antibodies by humanizing only those amino acids that are accessible at the surface of the V-regions of the recombinant F.sub.V. The resurfacing of murine monoclonal antibodies to reduce their immunogenicity could be beneficial in maintaining the avidity of the original monoclonal antibody in the reshaped version, because the natural framework-CDR interactions are retained. The value of maintaining the integrity of the framework-CDR interactions has been illustrated as summarized below.
In a recent research report, two different reshaped versions of the rat monoclonal antibody, Campath-9 (anti-human CD4), were generated (Gorman, S. D., Clark, M. R., Routledge, E. G., Cobbold, S. P. and Waldmann, H. (1991), Proc. Natl. Acad. Sci. U.S.A. 88, pp. 4181-4185). In one version, pV.sub.H NEW/C.sub.G1, the acceptor V.sub.H framework was from the human NEW-based heavy chain, which has 47% identical residues to the Campath-9 V.sub.H. While in the second version, pV.sub.H KOL/C.sub.G1, the acceptor V.sub.H framework was from the human KOL antibody, which has 72% identical residues to Campath-9 V.sub.H. Each reshaped antibody contained the identical V.sub.L domain from the human REI antibody sequence. However, the recombinant product of pV.sub.H KOL/C.sub.G1 had an avidity for CD4 that was substantially greater than the product of pV.sub.H NEW/C.sub.G1. The authors proposed a reshaping strategy where human sequences, that are highly homologous to the rodent antibody of interest, are transferred, by in vitro mutagenesis, into the rodent V-region to create a "bestfit" reshaped antibody. This strategy uses the term "bestfit" to describe the modeling process, however, there is no quantitative formula employed to assess "bestfit", and so in effect, the process is subjective. Additionally, there is no resurfacing concept presented in that paper.
The concept of reducing rodent-derived antibody immunogenicity through the replacement of exposed residues in the antibody framework regions which differ from those of human origin is discussed in a recent paper (Padlan, E. A. (1991), Molecular Immunology 28, pp. 489-498). In that paper, the variable domains of two antibody structures, KOL (human) and J539 (mouse), are examined. The crystal structures of the Fab fragments of these two antibodies have been elucidated to high resolution. The solvent accessibility of the exposed framework residues in the variable domains of these two antibodies were compared to a sequence database of human and murine antibody V-region subgroups. On the basis of his findings, Padlan proposed to reduce the antigenicity of allogeneic variable domains [murine V-regions], through replacement of the exposed residues in the framework regions with residues usually found in human antibodies. In murine sequences with the highest similarity to a given human sequence, the number of changes necessary to "humanize" a murine V-region surface would range from 6-15 amino acid changes per V-region. This reference suggests how to convert one antibody surface into another but no general method is developed. Application of the procedure is provided by two examples, a worst-case and a best-case.
Worst Case:
Among the representative murine kappa V.sub.L sequences examined for which its autologous V.sub.H has been sequenced, S107V.sub.L has the most residues that need to be replaced to humanize it. S107V.sub.L is most similar to the members of the human subgroup VKIV and JK2. The exposed or partially exposed residues that need to be replaced are those at positions 9, 10, 14, 15, 16, 17, 18, 22, 41, 63, 80, 85, 85, 100 and 106. Murine V-region S107V.sub.H is most similar in its framework to the members of the human subgroup VHIII and JH6. The exposed or partially exposed residues in S107V.sub.H that need to be replaced are those at positions 3, 40, 68, 73, 75, 76, 82b and 89. A total of 23 residues need to be replaced to humanize the variable domains of S107.
Best Case:
Among the murine V.sub.H sequences examined for which the autologous V.sub.L has also been sequenced, MOPC21V.sub.H has the least number of residues that need to be replaced to humanize it. MOPC21V.sub.H is most similar in its framework to the members of the human subgroup HIII and JH6. The exposed or partially exposed residues that need to be replaced are those at positions 1, 42, 74, 82a, 84, 89 and 108. MOPC21V.sub.L is most similar in its framework to human subgroup VKIV and JK4. The exposed or partially exposed residues that need to be replaced are those at positions 1, 9, 12, 15, 22, 41, 63, 68, 83 and 85. A total of 17 amino acids need to be replaced to humanize the variable domains of MOPC21.
Of the light chains in the Best- and Worst-Case examples cited above, S107V.sub.L required changes at 15 positions and MOPC21V.sub.L required changes at 10 positions. Only seven of the changes are common to both of these light chain sequences (see underlined residues). Moreover, of the heavy chain residues that need to be replaced to humanize the respective V-regions, S107V.sub.H required changes at 8 positions and MOPC21V.sub.H required changes at 7 positions. In this instance, only one position is common to both of these heavy chain sequences (see residues in boldface).
An analysis of S107 V-regions alone would not have led to the prediction of which residues to change in MOPC21. The reason for this is that the surface residues in Padlan's analysis are only determined by reference to the crystal structure analysis of one antibody. In addition, the basis for defining the surface exposure of an amino acid at a particular position on that crystal structure is a continuous gradient of change, e.g., the fractional solvent accessibility values (Padlan, E. A. (1990), Molecular Immunology 28, pp. 489-498) were computed, where: 0 to 0.2=completely buried, 0.2 to 0.4=mostly buried, 0.4 to 0.6=partly buried/partly exposed, 0.6 to 0.8=mostly exposed, and 0.8 or above=completely exposed. By limiting the analysis of exposed surface residues to a single crystal structure and by superimposing a broad range of solvent accessibility ratios on exposed residues, such a modeling strategy could be expected to have a wide margin of error in its calculations. This model fails to take into account the great majority of structural information available in the database for other antibody crystal structures.